Measuring the Affinities of RNA and DNA Aptamers with DNA Origami-Based Chiral Plasmonic Probes

Reliable characterization of binding affinities is crucial for selected aptamers. However, the limited repertoire of universal approaches to obtain the dissociation constant (KD) values often hinders the further development of aptamer-based applications. Herein, we present a competitive hybridization-based strategy to characterize aptamers using DNA origami-based chiral plasmonic assemblies as optical reporters. We incorporated aptamers and partial complementary strands blocking different regions of the aptamers into the reporters and measured the kinetic behaviors of the target binding to gain insights on aptamers’ functional domains. We introduced a reference analyte and developed a thermodynamic model to obtain a relative dissociation constant of an aptamer–target pair. With this approach, we characterized RNA and DNA aptamers binding to small molecules with low and high affinities.


Materials
DNA scaffold strands (p7560) were purchased from tilibit nanosystems; core staple strands (SI Table S1) from ThermoFisher; thiol modified DNA strands from Biomers; other DNA strands from IDT. Buffers and chemicals were purchased from Fisher Scientific or Sigma-Aldrich unless specified. All reagents were commercially available and used without any further purification. Type I ultrapure deionized (DI) water from the Milli-Q system was used for all experiments. Spin filters for DNA origami purification (cut off size 100 kDa) were manufactured by Millipore.     TTT ACG ACC GTG TGT GTT  GCT CTG TAA CAG TGT CCA TTG TCG T   3'Glucose aptamer  DNA  NA  40+16  ACG ACC GTG TGT GTT GCT CTG TAA CAG TGT  CCA TTG TCG T TTT AGAGTG

DNA origami assembly:
Core staples were mixed with docking staples at 1:1.5 ratio to obtain the staple solutions. For 1-step method, the oligonucleotides of interests (ONI) were added with the 2 times concentration of core staples. Scaffold strand (10 nM) and staples were mixed at 1:10 ratio in DNA origami folding buffer and then assembled using the established protocol by thermal annealing from 80˚C to 20˚C. 4,5 The DNA origami solution was then purified in wash buffer using spin filters (cut off size 100 kDa) for 3 times to remove the free staple strands following the protocol provided by the manufacture.

Gold nanorods (AuNRs) functionalization:
The AuNRs were synthesized using the previously published protocol 5 and functionalized with 5'-thiol-TTTTTT TTTTTT T-3' DNA strands by freeze-thaw method 6 . Bare AuNRs were mixed with thiol-polyT DNA strands at 1:10000 ratio. The mixture was supplemented with 0.05% SDS and frozen for 1h at -20˚C. The AuNR-DNA were washed by centrifugation at 7000 rcf for 30min in wash buffer for 4 times to remove free thiol-polyT DNA strands.

DNA origami-AuNRs assembly:
The DNA origami and AuNR-DNA were mixed at 1:15 ratio and annealed from 40˚C to 20˚C in assembly buffer. The DNA origami-AuNRs with free AuNR-DNA was either directly used for the next step or purified through gel electrophoresis in 0.7% agarose gel for 3h at 80 V.

Incorporation of oligonucleotides of interest (ONI):
The concentration of purified DNA origami-AuNRs was calculated by measuring its absorption value (peak ~650 nm) and dividing by the estimated extinction coefficient of 3.4 nM -1 ⋅cm -1 . The concentration of unpurified DNA origami-AuNRs was assumed to be equal to the input DNA origami concentration as the product output yield was generally very high. The DNA origami-AuNRs were mixed with the ONI in incorporation buffer. The ratio of ONI (comprised of the aptamer and the complementary strand, sequences in SI Tables S4, S6) and the purified DNA origami-AuNRs (~0.3 nM) or non-purified DNA origami-AuNRs (~0.5 nM) was varied between

Sample preparation for measuring circular dichroism (CD) signal:
To compare different DNA origami-AuNRs samples fabricated with different workflows, 3 completely independent experiments were conducted, during which DNA origami folding, purification, DNA functionalization of AuNRs, and the DNA origami-AuNRs assembly and purification were all performed separately.
For kinetic experiments, 7 µL solutions of the ONI-DNA origami-AuNRs probes were added to the 63 µL reaction buffer containing reference analyte or target and mixed by vortex. The 70 µL samples were immediately pipetted into the cuvette (within 30 seconds) and the CD amplitude at 620 nm was recorded with the Jasco J-1500 CD spectrometer. The CD amplitude of the aliquot ONI-DNA origami-AuNRs sample that underwent the same operation in the reaction buffer without target or reference analyte was used as the control points before target binding.
For thermodynamic experiments, the samples of ONI-DNA origami-AuNRs were incubated in 70 µL reaction buffer containing different concentration of reference analyte or target at room temperature with shaking at 100 rpm. The CD spectra were measured after overnight incubation.

Design benefits
As  Figure S3. The secondary structure of the Apt2 with the tail added to 5' end (left) and the CS2 with the tail added to the 3' end (right), generated by NUPACK. 12 Figure S4. (A, B) The TEM image of the probes of DNA origami-AuNRs after annealing (A) and room temperature (RT) incubation (B). Scale bar represents 100 nm. (C) The CD spectra of the reporter after annealing and room temperature incubation without ONI. Figure S5. (A, B) The CD spectra of purified probe of DNA origami-AuNRs incorporated by the Apt1R and CSR1 (9nt). The effects of the temperature (A) (the concentration ratio of ONI and reporter was fixed at 50:1) and the concentration ratio of the ONI and the DNA origami (B) (with annealing treatment). Figure S6. (A, B) The CD spectra of unpurified probes of DNA origami-AuNRs incorporated by the ONI-1. The effects of the temperature (A) (the concentration ratio of ONI and reporter was fixed at 50:1) and the concentration ratio of the ONI and the DNA origami (B) (with annealing treatment). (C) The comparison of ratio effects between the purified and unpurified samples. Figure S7. The secondary structure of the glucose DNA aptamer with the tail added to the 5' end (left) and the 3' end (right), generated by NUPACK. 12 Figure S8. The secondary structure of the ATP RNA aptamer with the tail added to the 5' end (left) and 3' end (right), generated by NUPACK (DNA tails were altered to RNA tails as chimera is unavailable). 12 S12 Figure S9. Workflow for measuring aptamer affinities.

Concentrations at equilibrium
In the samples without reference analytes or targets, the reaction 1 is at equilibrium; in the samples with reference analytes, reaction 1, 2, and 3 are at equilibrium; in the samples with targets, reaction 1, 4, 5 are at equilibrium.
Substitute the concentration into the equations S1 and S2. Therefore, Solve , , ′, , and ′: (Eq. S12) • • (Eq. S13) We define as the ratio of the AC concentrations in the presence and absence of the reference analyte; ′ as the ratio of the AC concentrations in the presence and absence of the target: (Eq. S17) ′ (Eq. S18) ( E q . S 1 9 ) where, , which is an unknown constant that requires manually adjust and treated as a free parameter.
Of note, the obtained values remained similar for a wide range of . Reasons and detailed examples are given below.

Circular dichroism signals at equilibrium
The measured circular dichroism signal (CD) at the minimum deep wavelength (~620nm), the measured absorption (Abs) at the maximum peak wavelength (~650 nm), and the normalized CD signal (N) can be expressed in terms of concentrations and molar optical coefficients as: is the molar extinction for the probe, which is irrelative to the configurational state.

The
, , and can also be written in terms of the fraction of probes in the closed configuration ( ). Thus, with the parameters: length as 0.34 nm/bp and diameter (d) as 2.3 nm to calculate the size of the components. 15 The distance (L) between the pivot point and the docking sites were approximately 26 nm. The distance (h) between the particle and the docking site is approximately 4.4 nm. The two bundles are linked with a single stranded spacer (8 nt) and we estimated the distance (p) between the two bundles as 2 nm (Figure S13). 3. Vary the concentration of the input reference analytes and measure CD and Abs. Obtain by fitting with using equation S17 by assigning a value to so the fitting is valid. The coefficient of determination R 2 indicates the goodness of the fitting. 4. Calculate with the obtained and using the equation S19. 5. Vary the concentration of the input target and obtain by fitting ′ with using equation S18 with the obtained . 6. Calculate as a ratio of with the obtained values of , , and to gain the relative dissociation constant compared to the reference analyte (equation S20).
workflow for DNA glucose aptamer: 1. The probe of DNA origami-AuNRs were incorporated with the glucose aptamer and its complementary strand (10 nt varied from 0.01 to 10. We estimate the value between 0.1-1 with the previous knowledge 16 and the variation of relative value was in an acceptable range. The fitting may fail when the value deviates from the real too much.